1. Field of the Invention
This invention relates to stabilized proteinases and methods for their manufacture and use. Specifically, this invention relates to proteinases that have been stabilized by admixture with water-soluble polymers or by conjugation of one or more strands of water-soluble polymer to sites on the proteinase.
2. Related Art
Proteinases are enzymes that are widely used in industrial genomics during the extraction and purification of DNA and RNA from cells and viruses. As used herein, “industrial genomics” refers to the commercial applications of DNA sequencing, fragment analysis and linear gene mapping. Examples of industrial genomics include the sequencing or diagnostic fragmentation of a portion of the DNA from human or animal cells for forensic purposes or to detect the presence of a gene associated with a malignancy or other disease. When nucleic acids are extracted from cells or tissues, the crude preparations include enzymes called nucleases that can catalyze the degradation of nucleic acids and thereby destroy the information encoded in their sequences. Classical methods for removing proteins, including nucleases, from preparations of nucleic acids include the precipitation of proteins by phenol, followed by precipitation of the nucleic acids by alcohol. Such methods do not adequately protect the nucleic acids from degradation.
More recent strategies for the purification of nucleic acids can involve the addition of proteinases to inactivate the nucleases and thereby protect the integrity of the nucleic acids (Strauss, W. M. (1998) in Ausubel, F. M., et al., (eds.) Current Protocols in Molecular Biology, Vol. 2, Unit 2.2, New York: John Wiley & Sons). The dissociation of tissues and the extraction of intact nucleic acids would be facilitated by the addition of chaotropic agents as well as proteinases, but the available proteinases are rapidly inactivated by the chaotropic agents. Proteinases (also referred to as proteases) catalyze the breakdown of proteins into fragments that include polypeptides, oligopeptides and amino acids. Certain proteinases (termed “endoproteinases”) cleave peptide bonds in the interior of the polypeptide chain, producing shorter peptide chains. Other proteinases (termed “exoproteinases”) cleave only one or a few residues at the amino or carboxyl terminal, thereby reducing the length of the protein or peptide substrate by one or a few residues in each cleavage cycle (Butler, M. J., et al., (1995) App Environ Microbiol 61:3145–3150; Lamango, N. S., et al., (1996) Biochem J 314:639–646).
In addition to their use in industrial genomics, proteinases can be used in the identification of pathology-related prions in the molecular diagnosis of transmissible spongiform encephalopathies such as scrapie, mad-cow disease and new variant Creutzfeldt Jakob Disease (McKinley, M. P., et al., (1983) Cell 35:57–62; Oesch, B., et al., (1994) Biochemistry 33:5926–5931; Caughey, B., et al., (1997) J Virol 71:4107–4110; Scott, M. R., et al., (1999) Proc Natl Acad Sci U S A 96:15137–15142).
The first step in the action of a proteinase involves the binding of its substrate to a specific domain of the enzyme. During or following such binding, a portion of the proteinase, herein termed the “active site” or “catalytic site,” interacts with the substrate to accelerate its cleavage. Proteinases with narrow specificities bind to and accelerate the digestion of a limited range of protein substrates. Other proteinases digest a wider variety of protein substrates. Insights into the binding of substrates and the catalytic properties of proteinases and other enzymes can be provided by analyses of the three-dimensional structures of their active conformations (Pähler, A., et al., (1984) EMBO J 3:1311–1314).
Since many applications of proteinases involve the cleavage of protein substrates in their interior, endoproteinases have wide utility in industry. Examples of endoproteinases include Pronase, an enzyme from Streptomyces griseus (EC 3.4.24.31), members of the family of bacterial subtilisin-like serine proteases (“subtilases”), such as subtilisin Carlsberg (EC 3.4.21.62), and three fungal enzymes (all from Tritirachium album): Proteinase K (EC 3.4.21.64), ProteinaseR and ProteinaseT (Samal, B. B., et al., (1991) Enzyme Microb Technol 13:66–70). The broad specificities of these enzymes, particularly the subtilases, make them valuable reagents in industrial genomics. The disadvantages of using these enzymes, however, include their vulnerability to self-digestion during storage and use, particularly at elevated temperatures (Bajorath, J., et al., (1988) Biochim Biophys Acta 954:176–182). Consequently, efforts have been made to increase their stability. U.S. Pat. No. 5,246,849 (Bryan, P. N., et al.); U.S. Pat. No. 5,278,062 (Samal, B. B., et al.); U.S. Pat. No. 5,837,517 (Sierkstra, L. N., et al.); U.S. Pat. No. 6,190,900 (Sierkstra, L. N., et al.); and U.S. Pat. No. 6,300,116 (Von der Osten, C. et al.) describe examples of proteinases the stability of which was improved by molecular and genetic techniques. Examples of chemically-derivatized proteases with enhanced performance for cleaning surfaces and fabrics are described by Olsen, A. A., et al., in U.S. Pat. Nos. 5,856,451; 5,981,718; 6,114,509 and 6,201,110 and by Bauditz, P., et al., in PCT publication WO 00/04138 A1 (pending as EP 1 098 964), each of which is incorporated herein fully by reference.
Unfortunately, most proteinases, including ProteinaseK and other subtilases, are susceptible to inactivation under conditions that are desirable for nucleic acid extraction for industrial applications, particularly as adapted to high throughput robotic instruments. Inactivation of proteinases is accelerated by exposure to heat and/or certain alterations in their ionic environment and by the presence of chaotropic agents, exemplified by guanidinium thiocyanate (“GdnSCN”), guanidinium hydrochloride, urea and anionic detergents such as lithium dodecyl sulfate or sodium dodecyl sulfate (“SDS”). Thus, there is a compelling need for proteinases that retain their activity under harsh conditions, such as the elevated temperatures and the presence of the denaturants that can be advantageously used in molecular diagnostics and industrial genomics.
Since the 1970s, attempts have been made to use the covalent attachment of polymers to improve the safety and efficacy of various enzymes for pharmaceutical use (Davis, F. F., et al., U.S. Pat. No. 4,179,337). Some examples include the coupling of poly(ethylene oxide) (“PEO”) or poly(ethylene glycol) (“PEG”) to adenosine deaminase (EC 3.5.4.4) for use in the treatment of severe combined immunodeficiency disease (Davis, S., et al., (1981) Clin Exp Immunol 46:649–652; Hershfield, M. S., et al, (1987) N Engl J Med 316:589–596). Other examples include the coupling of PEG to superoxide dismutase (EC 1.15.1.1) for the treatment of inflammatory conditions (Saifer, M., et al., U.S. Pat. Nos. 5,080,891 and 5,468,478) and to urate oxidase (EC 1.7.3.3) for the elimination of excess uric acid from the blood and urine (Inada, Y., Japanese Patent Application No. 55-099189; Williams, L. D., et al., PCT publication WO 00/07629 A3; Kelly, S. J., et al., (2001) J Am Soc Nephrol 12:1001–1009; Sherman, M. R., et al., (2001) PCT publication WO 01/59078 A2). Such polymer-conjugated enzymes, which were designed for therapeutic use, do not need to retain their activities under the harsh conditions used in industrial applications.
Poly(ethylene oxide) and poly(ethylene glycol) are polymers composed of covalently linked ethylene oxide units. These polymers have the general structureR1—(OCH2CH2)n—R2where R2 may be a hydroxyl group (or a reactive derivative thereof) and R1 may be hydrogen (as in PEG diol), a methyl group (as in monomethoxy PEG) or another lower alkyl group (e.g., as in iso-propoxy PEG or t-butoxy PEG). The parameter n in the general structure of PEG indicates the number of ethylene oxide units in the polymer and is referred to herein as the “degree of polymerization.” PEGs and PEOs can be linear, branched (Fuke, I., et al., (1994) J Control Release 30:27–34) or star-shaped (Merrill, E. W., (1993) J Biomater Sci Polym Ed 5:1–11). PEGs and PEOs are amphipathic, i.e. they are soluble in water and in certain organic solvents and they can adhere to lipid-containing materials, including enveloped viruses and the membranes of animal and bacterial cells. The covalent attachment of PEG to a protein or other substrate is referred to herein as “PEGylation.” Certain random or block or alternating copolymers of ethylene oxide (OCH2CH2) and propylene oxide (shown below)
have properties that are sufficiently similar to those of PEG that these copolymers can be substituted for PEG in many applications (Hiratani, H., U.S. Pat. No. 4,609,546; Saifer, M., et al., U.S. Pat. No. 5,283,317). The term “poly(alkylene oxides)” and the abbreviation “PAOs” are used herein to refer to such copolymers, as well as to poly(ethylene glycol) or poly(ethylene oxide) and poly(oxyethylene-oxymethylene) copolymers (Pitt, C. G., et al., U.S. Pat. No. 5,476,653).
Certain polymer-modified proteinases in the prior art are claimed to have improved properties for use in laundry detergents and other washing applications (Bauditz, P., et al. and Olsen, A. A., et al., supra). However, many preparations of proteinases that are currently available contain significant quantities of impurities, including fragments of the proteinase itself, as well as unrelated contaminating proteins. Moreover, the polymer-conjugated proteinases of the prior art may contain variable amounts of both free and attached polymer, which amounts may be unknown to or inadequately described by their producers. As a result, the properties of the available polymer-coupled proteinases are variable and unpredictable, making them unsuitable for use in quantitative industrial applications. Certain polymer-conjugated proteinases of the prior art contain an unstable linkage between the polymer and the enzyme (e.g., those described in Harris, J. M., U.S. Pat. No. 6,214,966). Such conjugates can deteriorate during storage, even at 0–8° C., and are especially labile under the conditions used in industrial applications. Thus, there is a need for proteinases that have longer storage lives and greater stability during use than those that are currently available.